MAGE proteins are normally expressed only in developing germ cells but are aberrantly expressed in some nevi, most melanomas, and many malignancies. Their selective expression makes them nearly tumor specific targets but the dominant paradigm regards MAGE expression as a functionally irrelevant byproduct of cellular transformation. This project challenges this paradigm by proposing that MAGE regulate cell growth and contribute to tumor development by regulating KAP1, a scaffolding protein that in turn mediates gene regulation by the large family of KRAB domain containing zinc finger transcription factors (KZNFs). The long term goals are: 1. To show how MAGE proteins contribute to cell growth and survival by functioning as master regulators of KZNFs, and 2. To develop novel therapies for malignancies based on interfering with MAGE expression or function. The Specific Aims are: Aim 1: To identify and manipulate MAGE effects on gene regulation in human melanocytes. To determine MAGE effects in melanocytes, we will correlate MAGE expression with genome wide KAP1 binding, chromatin compaction, and gene repression using ChIP-seq and RNA-seq of normal human melanocytes, with and without ectopic MAGE expression. Controls will use low passage human melanoma cell lines, with and without shRNA mediated MAGE knockdown, and chromatin from melanocytic nevi removed for cosmetic purposes. Analysis will include comprehensive gene annotation, clustering, and network analysis for multi-parametric identification of global effects of MAGE on cellular processes and critical downstream components. Validation will test the role of downstream network components with assays for proliferation, survival and cell cycle regulation, by selective inhibition of pathway components with RNAi. To begin to develop and validate small molecules that inhibit MAGE effects, MAGE knockdown effects on KAP1 binding and gene expression profiles will be compared to the effects of small molecule MAGE inhibitors. Aim 2: To determine mechanisms for differential MAGE effects on different genes. We have discovered that MAGE proteins can enhance or decrease KZNF and KAP1 mediated repression of specific genes. To determine mechanisms for differential MAGE effects on gene regulation, we will test the hypothesis that differences in the composition of KRAB domains determines whether MAGE expression increases, decreases, or has no effect on KZNF mediated gene repression. These experiments will test MAGE effects with a comprehensive series of KRAB domains for: a. KAP1- KRAB binding in a mammalian two hybrid assay, b. KAP1 mediated gene repression in a luciferase reporter gene assay, and c. KAP1 mediated KZNF ubiquitination by immunoblotting.